Intrinsic and extrinsic regulation of haematopoiesis by retinoic acid receptor gamma

© 2015 Dr. Chacko JosephRetinoids regulate numerous body functions including haematopoiesis by binding to nuclear heterodimers of retinoic acid receptors (RARs) and retinoid X receptors (RXRs).RARs have been reported to regulate haematopoiesis thorough cell intrinsic and extrinsic mechanisms. The aim of this thesis was to obtain a greater understanding of RAR-mediated regulation of intrinsic and extrinsic haematopoiesis, particularly focusing on RARγ. The RAR subtype Rarγ was differentially expressed among haematopoietic stem and progenitor cells (HSPCs) with the highest expression restricted to the most primitive long-term haematopoietic stem cells (HSCs). The pan-RAR agonist, all-trans retinoic acid (ATRA) is able to increase the number of murine HSCs in vitro, however, this findin g has been difficult to translate into human HSCs, likely due to the different effects of the RARs in haematopoiesis. We have established an in vitro serum-free culture condition to aid the study of the effects of RAR ligands on murine HSPCs, which may help to improve human HSPC studies in future. Short-term RAR antagonism has been shown to increase granulocyte numbers in mice. However, our studies showed that short-term in vivo pan-RAR antagonism in mice showed no effect in accelerating granulocyte recovery following bone marrow transplantation. Hoxa1, an early target gene of RARγ, has poorly defined roles in haematopoiesis. It exists as multiple isoforms generated by alternative splicing; the DNA binding homeobox domain containing Hoxa1 spliceform (Hoxa1-FL) and the homeobox lackingHoxa1 spliceform (Hoxa1-T), which were found to be differentially expressed in murine HSPCs. Hoxa1 spliceforms displayed opposing effects on the in vitro cell proliferation and colony forming capacity of immature haematopoietic cells. Mice transplanted with immature haematopoietic cells overexpressing both Hoxa1 spliceforms (WT-Hoxa1)developed clinical features of myelodysplastic syndromes (MDS). These included peripheral blood (PB) leukopenia, reduced haemoglobin levels and ineffective bone marrow (BM) haematopoiesis, but complete absence of transformation to secondary acute myeloid leukaemia (sAML). In contrast, overexpression of the Hoxa1-FL isoform alone (MUT-Hoxa1) resulted in a more aggressive MDS that spontaneously progressed to sAML. DNA damage repair-related pathways were deregulated exclusively in the MUT-Hoxa1 overexpressing megakaryocyte erythroid progenitors compared to those overexpressing WT-Hoxa1. Of importance, HOXA1 was overexpressed in approximately 50% of human MDS patients. As such, these twoHoxa1 mouse models, developed by altering Hoxa1 spliceform expression, will be highly valuable in identifying better therapies for a significant subset of MDS patients. Deletion of Rarγ in either osterix-targeted osteoprogenitor cells or nestin-targeted cells was performed to elucidate the extrinsic cell specific contribution of RARγ towards haematopoiesis. Osteoprogenitor specific deletion of Rarγ had no impact on haematopoiesis. In contrast, nestin-targeted deletion of Rarγ resulted in altered lymphopoiesis, as indicated by a smaller thymus and reductions in BM PreB cells and thymic double negative 4 (DN4) T lymphocytes. Moreover, short-term in vivo activation of pan-RAR signaling by ATRA treatment increased BM PreB cells and DN4 Tlymphocytes. Furthermore, nestin-expressing cells were identified within the thymic microenviromment, restricted to a subpopulation of neural crest (NC) mesenchymal cells. These NC mesenchymal cells expressed high mRNA levels of Rars, Cxcl12 and Scf. These studies show that nestin-targeted cells in the BM and thymic microenvironments are regulators of B and T lymphopoiesis, respectively, and that RARγ regulates lymphocyte numbers through these nestin-targeted microenvironments. Collectively, the results presented in this thesis help to further delineate the roles of RARγ in the intrinsic and extrinsic regulation of haematopoiesis